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Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102-104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns., Competing Interests: The authors have declared that no competing interests exist.

Mycoplasma synoviae is one of the economically most significant avian Mycoplasma species. It can cause great financial losses to the poultry industry by inducing respiratory diseases, infectious synovitis, or eggshell apex abnormalities. There are different approaches to control M. synoviae infection. Although antimicrobial therapy cannot replace long-term solutions, like eradication and vaccination, this strategy can be effective in the short term, as adequate antibiotic treatment can relieve economic losses through the attenuation of clinical signs and reduction of transmission. Using broth microdilution method, minimal inhibitory concentration (MIC) values to fourteen antibiotics related to eight antimicrobial groups were determined in 96 M. synoviae strains. Whole genome sequencing and sequence analysis revealed mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin. Molecular markers responsible for the high MICs to fluoroquinolones were found in the gyrA, gyrB, parC and parE genes. Besides, single nucleotide polymorphisms identified in genes encoding the 23S rRNA were found to be responsible for high MICs to the 50S inhibitor macrolides and lincomycin, while amino acid change in the 50S ribosomal protein L22 could be associated with decreased susceptibility to macrolides. The revealed mutations can contribute to the extension of knowledge about the genetic background of antibiotic resistance in M. synoviae. Moreover, the explored potentially resistance-related mutations may serve as targets for molecular biological assays providing data of antibiotic susceptibility prior to the laborious and time-consuming isolation of M. synoviae strains., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mycoplasma synoviae (MS) is a highly prevalent bacterial species in poultry causing disease and severe economic losses. Antibiotic treatment is one of the control strategies that can be applied to contain clinical outbreaks in MS-free flocks, especially because this bacterium can be transmitted in ovo. It becomes, then, very important for veterinarians to know the antibiotic susceptibility of the circulating strains in order to choose the most appropriate first-line antibiotic molecule as a proactive role in fighting antibiotic resistance. We evaluated the Minimum Inhibitory Concentrations (MICs) of enrofloxacin, oxytetracycline, doxycycline, erythromycin, tylosin, tilmicosin, spiramycin, tiamulin, florfenicol and lincomycin for MS isolates collected between 2012 and 2017 in Italy. A total of 154 MS isolates from different poultry commercial categories (broiler, layer, and turkey sectors) was tested using commercial MIC plates. All MS isolates showed very high MIC values of erythromycin (MIC90 ≥8 μg/mL) and enrofloxacin (MIC90 ≥16 μg/mL). MIC values of doxycycline and oxytetracycline obtained were superimposable to each other with only a one-fold dilution difference. Discrepancies between MIC values of tylosin and tilmicosin were observed. Interestingly, seven isolates showed very high MIC values of lincomycin and tilmicosin, but not all of them showed very high MIC values of tylosin. Most of the MS isolates showed low MIC values of spiramycin, but seven strains showed a MIC ≥16 μg/mL. In the observation period, the frequency of the different MIC classes varied dependently on the tested antibiotic. Interestingly, tilmicosin MICs clearly showed a time-dependent progressive shift towards high-concentration classes, indicative of an on-going selection process among MS isolates. Until standardized breakpoints become available to facilitate data interpretation, it will be fundamental to continue studying MIC value fluctuations in the meantime in order to create a significant database that would facilitate veterinarians in selecting the proper drug for treating this impactful Mycoplasma., Competing Interests: The authors have declared that no competing interests exist.

Mycoplasma bovis is an important respiratory pathogen of cattle across Europe and is included in the MycoPath pan-European antimicrobial susceptibility monitoring programme. M. bovis strains (232) were isolated from cattle, not recently treated with antimicrobials, at diverse geographical locations in France, Great Britain, Hungary, Italy and Spain during 2014 to 2016. Only one isolate per farm and per outbreak was retained. For each isolate, the MICs of ten antimicrobials were determined in a central laboratory using a broth microdilution method with modified Eaton's medium and incubation at 35 °C ± 1 °C for 24 ± 6 h. MIC 50 /MIC 90 (mg/L) values for the 232 strains were: danofloxacin 0.25/1; enrofloxacin 0.5/8; marbofloxacin 1/4; gamithromycin >64/>64; spiramycin 8/16; tilmicosin >64/>64; tulathromycin >64/>64; tylosin 64/>64; florfenicol 4/8; oxytetracycline 8/32. Minor between-country differences in the MIC 90 values were observed for the fluoroquinolones, spiramycin and oxytetracycline, whilst the MIC values for the other compounds were similar. Spain and Italy had the higher MIC 90 values for the fluoroquinolones. Compared with the 2010-2012 study (156 isolates) results are similar, with an overall MIC 50 increase of at most one doubling dilution for enrofloxacin, spiramycin, tylosin, florfenicol and oxytetracycline. In contrast, the MIC 90 value for oxytetracycline decreased from >64 to 32 mg/L. Standardized laboratory methods and interpretive criteria for MIC testing of veterinary mycoplasmas are clearly needed; there are currently no clinical breakpoints available to facilitate data interpretation and correlation of MICs with in vivo efficacy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Silver nanoparticles (AgNPs) are of interest due to their antimicrobial activity and are seen as potential candidates to replace antibiotics in animal husbandry. A few studies have focused on this new application, but they lack any considerations about residual accumulation of AgNPs in edible animal tissues and animal products. In this research, a 22 day in vivo study was carried out by oral administration of 20 nm spherical PVP coated AgNPs to hens. Six doses of approximately 1 mg kg -1 of AgNPs-PVP each were administered to animals throughout the experimentation. Atomic absorption spectroscopy (AAS) was used for quantitative determination of residual total Ag in different organs and matrices. The analyses showed that Ag accumulates in livers (concentration ranging from 141 μg kg -1 to 269 μg kg -1 ) and yolks (concentration ranging from 20 μg kg -1 to 49 μg kg -1 ) but not in muscles, kidneys, and albumen belonging to hens of the treated group (tG2). Ag was not detected in animals of the control group (uG1) (i.e., total Ag < LOD = 10 μg kg -1 ). Single particle inductively coupled plasma mass spectrometry (spICP-MS) and scanning electron microscopy with energy dispersive X-ray detection (SEM-EDX) were employed to elucidate the presence of AgNPs in livers and yolks belonging to tG2 animals. spICP-MS highlighted that part of residual Ag found in livers (about 5-20%) is in NP form with an average dimension of approximately 20 nm. SEM-EDX technique confirmed the presence of AgNPs only in livers of treated animals. The results show that feeding AgNPs to hens may become a source of consumer exposure to AgNPs. As far as we know this is the first study showing transfer of AgNPs or reaction products thereof from animal feed to animal products.

A 55 g, 7 mo old Chinese three-striped box turtle ( Cuora trifasciata ) was presented with a 2 wk history of lethargy and anorexia. The owner attempted various antibiotic treatments without clinical improvement. A cystoscopic evaluation of the coelom was performed. The liver appeared dark red-to-brown presenting multifocal irregularly shaped white-to-tan and roughly round areas of discoloration on the capsular surface. An adult keeled box turtle (Pyxidea mouhotii), which died in the same facility at the same time, had similar hepatic lesions detected upon necropsy. From hepatic lesions, an antibiotic-resistant Escherichia coli was isolated. Antibiotic treatment based on sensitivity testing led to a rapid resolution of the clinical signs in the Cuora trifasciata , with gain of appetite 4 days after the first antibiotic administration. A recheck cystoscopy demonstrated macroscopic resolution of hepatic lesions. This report demonstrates, to the best of the authors' knowledge, for the first time the clinical usefulness of cystoscopy for the diagnostic evaluation of the coelom of small chelonians. This minimally invasive technique is a valuable clinical tool in the medical approach to posthatchling chelonians.

The mechanism responsible for acquired decreased susceptibility to macrolides (14-membered erythromycin [Ery], 16-membered tylosin [Ty] and tilmicosin [Tm]) and to lincosamides (lincomycin [Ln]) was investigated in Mycoplasma synoviae, a pathogen that causes respiratory infections and synovitis in chicken and turkey. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 49 M. synoviae isolates, M. synoviae type strain WVU1853, and reference strain FMT showing minimal inhibitory concentrations (MICs) to Ty (≤ 0.015 to 2 μg/ml), Tm (0.03 to ≥ 8 μg/ml), and Ln (0.125 to 8 μg/ml); MICs to Ery ranged from 32 to ≥ 128 μg/ml. Our results showed that the nucleotide substitution G748A (Escherichia coli numbering) in domain II of one or both 23S rRNA alleles may account for a slight increase in MICs to Ty and Tm (up to 0.5 and 2 μg/ml, respectively). No correlation between the presence of G748A and decreased susceptibility to Ln was found. However, the presence of the point mutations A2058G or A2059G in domain V of one or both alleles of the 23S rRNAs was correlated with a more significant decrease in susceptibility to Ty (1-2 μg/ml), Tm (≥ 8 μg/ml), and Ln (≥ 8 μg/ml). All M. synoviae isolates tested had a G2057A transition in the 23S rRNAs consistent with previously described intrinsic resistance to Ery. Mutations G64E (one isolate) and Q90K/H (two isolates) were identified in the L4 and L22 proteins, respectively, but their impact on decreased susceptibility to macrolides and lincomycin was not clear.

The in vitro activity of enrofloxacin against 73 Mycoplasma synoviae field strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 μg/ml. The estimated MIC50 and MIC90 values for enrofloxacin were 2 and 8 μg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) of M. synoviae have MICs of ≥ 2 μg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) in M. synoviae isolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥ 1 μg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison of vlhA molecular profiles revealed the presence of 9 different genotypes in the Israeli M. synoviae field isolates and 10 genotypes in the European isolates; only one vlhA genotype (type 4) was identified in both cohorts. Based on results of vlhA molecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistant M. synoviae isolates are suggested.

Trichilia emetica Vahl. (Meliaceae) is a tree widely distributed in Tropical Africa. It has been used in Mali folk medicine for the treatment of various illnesses. The aim of this work was to study the hepatoprotective and antibacterial effects of a crude aqueous extract from Trichilia emetica root. An ethyl ether fraction from the aqueous extract was also prepared and studied. We have examined the hepatoprotective activity of the extracts on CCl4-induced damage in rat hepatocytes, their toxicity using the brine shrimp bioassay and their antibacterial activity against clinical isolated bacterial strains, which are commonly responsible for respiratory infections. A preliminary phytochemical analysis showed a high polyphenolic content in the aqueous extract and the presence of limonoids in the ethyl ether fraction. These latter compounds may be considered responsible for the good activity against the bacterial strains tested. Trichilia emetica extracts exerted also a significant (P<0.05) hepatoprotective effect at a dose of 1000 microg/ml both on plasma membrane and mitochondrial function as compared to silymarin used as a positive control. These activities may be a result of the presence of either polyphenols or limonoids. Finally, both the aqueous extract and its ethyl ether fraction did not show toxicity (LC50>1000 microg/ml) in the brine shrimp bioassay.

Clinical efficacy and tolerability of two different regimens of cefaclor were studied in 410 paediatric patients affected by non-recurrent acute otitis media (AOM). 204 patients were randomly treated with cefaclor given at the dosage of 50 mg/kg/die in two doses for 5 days; 206 patients with 40 mg/kg/die in two doses for 10 days. Diagnosis of acute otitis media was based on the following signs and symptoms: otalgia, fever, possible otorrhea, alterations of tympanic membrane under otoscopic examination. At the end of the treatment 95.5% of patients were cured after short-term therapy and 94.8% after long-term therapy. At the follow-up (two months after the interruption of treatment) 19 and 17 patients respectively had a recurrence. Side effects were observed in 6.9% and 8.7% of patients respectively (not statistically significant); they were mild, never requiring interruption of the treatment. Cefaclor therapy is very effective for treating children aged 2-6 years affected by acute otitis media, with no significant difference related to treatment duration. Short-course treatments appear better in terms of compliance and tolerability.

Nasopharyngeal swabs were collected from children aged 3-5 years in central Italy who were attending day-care centres or hospital outpatient clinics. One hundred and twenty-one strains of Streptococcus pneumoniae isolated were tested for susceptibility to penicillin, cefotaxime, erythromycin, clindamycin, tetracycline, chloramphenicol and cotrimoxazole. A high prevalence of penicillin-resistant (14%), erythromycin-resistant (60%) and multiply resistant strains (53%) were found. An unusual finding was that 49 of the 64 (76.6%) multiply resistant strains were penicillin-susceptible, 28 serogroup 6 strains also being resistant to the other antibiotics tested. Such strains have not previously been reported from Italy but have the same features as strains recently found in child carriers in the eastern Mediterranean area.

Objectives: To evaluate the incidence of bacterial colonization in the throat and in urines of children admitted to a paediatric ward in the year 1994. To test the sensitivity of isolates on the most common antibiotics used in therapy., Methods: The investigation was carried out on a group of 270 children (125 male and 145 female), aged between 3 months and 12 years, hospitalized with feverish infectious pathology in the department of infectious and Tropical Diseases of the University "La Sapienza" of Rome. The cultures of the throat swabs and on urines were performed on the admission of the children before the beginning of the therapy., Results: The throat-swab cultures showed pathogenous microrganisms in 232 samples (85.9%) with a slight prevalence of Gram-negative bacteria (122) with respect to Gram-positive (110) and saprophytic microbial flora (38). The urine cultures proved to be positive in 81 cases (30%) with a prevalence of Gram-negative (56) above Gram-positive isolates (25)., Conclusions: The two/thirds of paediatric patients hospitalized in an Infectious Diseases Department appeared to be colonized in the upper respiratory tract, whereas in about 10% of them a marked bacteriuria was clearly evident, often in the absence of specific symptoms. A few isolates either from the throat or from urines, showed resistance to the common antibacterial agents.

The in vitro activity of elsamicin A (ELS) was investigated compared with that of doxorubicin (DX) on two sensitive breast cancer cell lines: one estrogen receptor-positive (ER+, MCF7) and one estrogen receptor-negative (ER-, MDA-MB-231) line, and on a DX-resistant subline (MCF7DX). The activity of the two drugs was also investigated on 19 clinical breast cancer specimens from untreated patients. The drugs were tested at pharamcologically relevant concentrations, as calculated from the area under the curve for a 3 h exposure to the lethal dose producing 10% mortality (LD10) in mice, and at 10- and 100-fold concentrations. In DX-sensitive lines, a greater inhibition of RNA and DNA precursor incorporation, as well as of cell proliferation, was caused by ELS than by DX. Moreover, the antiproliferative effect was 10-fold higher in the ER+ MCF7 than in the ER- MDA-MB-231 cell line (IC50: 0.25 versus 0.21 micrograms/ml). ELS was cross-resistant to DX in the MCF7DX subline. In clinical specimens, effects on DNA precursor incorporation were more often observed for ELS than for DX at the same drug concentrations. The in vitro sensitivity to ELS was more pronounced for ER+ than for ER- tumors: minimal inhibiting concentrations of the drug were 0.1 and 3.5 micrograms/ml, respectively, in the two groups. If confirmed in a larger series of human breast tumors, these in vitro results would indicate a promising role for ELS in clinical treatment, mainly of ER+ breast cancer patients.

The authors have evaluated the efficacy and tolerance of teicoplanin against Gram-positive infections in 8 patients (four with endocarditis, one with cholangitis, one with purulent arthritis, one with gathered fistula and one with general pyoderma), arose both in the immunocompetent and immunodepressed subjects. All patients received IM teicoplanin at a dosage of 200-400 mg/die, according to the severity of the infectious process. Teicoplanin proved to be an effective and well-tolerated drug in the treatment of staphylococcus infections, both for immunocompetent and with serious deficit of the immune system subjects.

An A-protein positive strain of Staphylococcus aureus was grown in varying concentrations of erythromycin, clindamycin and miocamycin at sub-MIC levels and incubated in purified human polymorphonuclear leukocytes (PMNs). The presence of A-protein produced resistance of the above strain to phagocytosis and killing by PMNs and a slight resistance to opsonization by normal serum. The addition of antibiotics, which inhibit protein biosynthesis, caused significant changes in the ability of this bacterium to resist opsonization by serum complement. The antimicrobial agents produced a significant enhancement of bacterial killing and phagocytosis. In particular, clindamycin and erythromycin, at sub-MIC concentrations, modified the hair-like structure of S. aureus, which contains A-protein, and were able to affect the interaction of the strain with phagocytic cells.